The ability to characterize cells by gene expression provides a wide variety of applications in therapy, diagnostics and bio/medical technology. However, in many of these applications, the starting or source material such as stem cells, cancerous cells, identified neurons, embryonic cells, etc. is highly limiting, making it necessary to amplify the targeted mRNA populations. Two existing methods for amplifying mRNA populations suffer from significant limitations. One method, the Brady and Iscove method (Brady et al., 1990, Methods Mol & Cell Biol 2, 17–25), produces only short (200–300 bp), extreme 3′ fragments of mRNAs using a PCR-based method which exponentially amplifies artifacts. A second method, the Eberwine protocol (Eberwine et al. (1992) Proc. Natl. Acad. Sci USA 89, 3010–3014) provides sequential linear amplification steps and is the current method of choice for amplifying mRNA populations from limiting material. Nevertheless, this protocol suffers from a number of deficiencies. For example, the amplified product does not represent full-length aRNA for many endogenous mRNAs, and hence the method is of limited use for generating probes or cDNA libraries.
There are two commonly used methods to analyze labeled mRNA populations. The first is to use microarray technology, in which PCR products are spotted at high density on microscope slides. This technology has an estimated sensitivity of 10 copies per cell. At this level, it would be barely possible to detect rare transcripts, if at all. This is due, in part, to the fact that rare transcripts will incorporate less fluorescent label than highly expressed transcripts, and will not be as bright. This same principle applies to the use of filter technology, in which cDNAs or PCR products are spotted onto a nylon membrane. This technology has an estimated sensitivity of ˜50 copies/cell and would clearly not detect any rare transcripts.
Normalization allows the detection of RNA transcripts expressed at low levels. This is important because ˜30% of all transcripts are present at 1–10 copies per cell (“rare” transcripts). Other genes are expressed at much higher levels, for example from 50 (moderately low) to 10,000 (high) copies per cell and up. Thus, if a population of poly(A) mRNA is labeled using reverse transcriptase, oligo dT, and radioactive/fluorescent nucleotides, the great majority of label will be incorporated into highly expressed transcripts. Normalization generates a reduction in the number of highly expressed transcripts relative to more rare transcripts. In an ideal case, a perfect normalization yields a single copy of every unique transcript in a population. As a result, all transcripts are equally labeled, and can be easily detected by either microarray or by filter.
Normalization/subtraction removes highly expressed “tester” transcripts that are in common with a “driver” population. The remaining, unhybridized sample are then labeled and analyzed. A significant limitation is that the subtracted product is often present in very low amounts. This makes experiments technically very difficult. Moreover, often times a single round of normalization is insufficient to either normalize or subtract two populations. Thus, the small amount of product that remains from the first normalization/subtraction then has to be used in a second, and sometimes a third, round to more fully subtract a population. By this time, very little product remains in the tester population, making it difficult to label and analyze experimentally. In order to get around this problem, PCR has been employed to amplify the differences that remained following a round of normalization/subtraction. This increases the amount of unhybridized tester to a level such that additional rounds of subtraction can be performed. Currently, three to four rounds of subtraction and PCR amplification are required to fully subtract two populations and identify unique transcripts in either pool. One problem with PCR is that some transcripts are preferentially amplified over others. Thus, in amplifying the differences left after a subtraction round, some will not be amplified, while others will be amplified at higher levels than others. This exaggerates the need for another round of subtraction.
The present invention provides the benefits of normalization without the limitations of PCR by combining a normalization protocol with a linear amplification protocol. By using an RNA polymerase like T7 to amplify the differences following a subtraction instead of PCR, the differences are linearly amplified. In addition, such polymerases do not show the same bias in amplifying transcripts that PCR does. As a result, it is easier to assure that what remains following a round of amplification will not be additionally biased by the subsequent amplification.
Relevant Literature
Sippel (1973) Eur. J. Biochem. 37, 31–40 discloses the characterization of an ATP:RNA adenyl transferase from E. coli and Wittmann et al. (1997) Biochim. Biophys. Acta 1350, 293–305 disclose the characterization of a mammalian poly(A) polymerase. Gething et al. (1980) Nature 287, 301–306 disclose the use of an ATP:RNA adenyltransferase to polyadenylate the '3 termini of total influenza virus RNA. Eberwine et al. (1996) U.S. Pat. No. 5,514,545 describes a method for characterizing single cells based on RNA amplification. Eberwine et al. (1992) Proc. Natl. Acad. Sci USA 89, 3010–3014, describe the analysis of gene expression in single live neurons. Van Gelder, et al. (1990) Proc Natl Acad Sci U S A.87(5):1663–7. describe amplified RNA synthesized from limited quantities of heterogeneous cDNA. Gubler U and Hoffman B J. (1983) Gene (2-3), 263–9, describe a method for generating cDNA libraries, see also the more recent reviews, Gubler (1987) Methods in Enzymology, 152, 325–329 and Gubler (1987) Methods in Enzymology, 152, 330–335. Clontech (Palo Alto, Calif.) produces a “Capfinder” cloning kit that uses “GGG” primers against nascent cDNAs capped with by reverse transcriptase, Clontechniques 11, 2–3 (October 1996), see also Maleszka et al. (1997) Gene 202, 39–43.
Copending U.S. Ser. No. 09/049,806 now, U.S. Pat. No. 6,114,152 and U.S. Ser. No. 09/566,570 describe methods, such as using polyadenyltransferase to add known 3′ sequences to aRNA molecules, which may be used in the subject methods.